A developing molar tooth germ is encapsulated within the jaw from which it will eventually erupt. The tooth germ is first observed as a developing bud (bud stage), which fans out into a cap-like structure (cap stage), and finally develops into a bell-like form (bell stage). It is during the late bell stage that odontoblasts and ameloblasts differentiate and deposit the organic matrices of dentin and enamel. It has been well established that development of the tooth germ depends on reciprocal interactions between the epithelial and mesenchymal tissues (reviewed in: Thesleff et al., 1991).
Previously, Baba et al., (1996) have shown that molar tooth germs isolated from 16.5-day mouse embryos can be dissociated by enzymatic treatment. When the epithelial cells were separated from the mesenchymal cells, neither secreted enamel proteins nor cell proliferation were observed in either of the cultures. However, intriguingly, when the dissociated cells were cultured together, secretion of enamel proteins and cell proliferation were observed. Furthermore, the dissociated cells self-assembled back into a morphologically correct tooth germ that was successfully cultured for more than 20 days. The authors hypothesized that since the tooth germ lacked a blood supply, its development was prematurely terminated.
Tissue engineering is an interdisciplinary field that has evolved from the combined expertise of life sciences and engineering principles for the creation of biological substitutes that maintain, restore, or improve tissue function (Kim et al., 1999). Several tissues such as liver, intestine, bone, and cartilage have been successfully engineered (Kim et al., 1999). Dissociated cells from a tissue or organ have been used to seed biodegradable polymer scaffolds, which are implanted within a suitable host such that a sufficient blood supply would allow the cells to organize into higher ordered structures around the scaffold. The maintenance of cell structures, such as those present in organs, is not possible without a blood supply. Within a matter of weeks the scaffold dissolves and the dissociated cells will have organized into a tissue or organ that was pre-determined by the size and shape of the original scaffold. Tissue resembling small intestine, consisting of a neomucosa lined with smooth muscle, columnar epithelium, and goblet cells having villus-like structures, have been generated using the above approach (Choi and Vacanti, 1997). Epithelial-mesenchymal cell interactions are as essential for developing teeth as they are for the proper development of intestinal tissues. In the tooth, mesenchymal cells form the dentin while cells of epithelial origin form the enamel. Although each mineralized tissue is formed from its respective cells of origin, epithelial-mesenchymal interactions are required to initiate the mineralization process.
The demonstrated establishment of bioengineered epithelial-mesenchymal cell-cell communications (intestine) and the synthesis of mineralized tissues (bone and cartilage) necessary for growing teeth have already been accomplished. A significant need exists for replacement teeth as observed from the common use of dental implants (year 2000 projected number of dental implant procedures was 910,000 with a compound annual growth rate of 18.6% from 1998 to 2005, (Annual Industry Report, 2000). A biological tooth substitute that is properly formed and integrated into the jaw of a human patient would outlast synthetic dental implants since a living tooth responds to its environment by migrating to maintain a proper bite, and has some regenerative properties in response to injury. Implants do not have these capabilities. In addition, people who have genetically inherited enamel (amelogenesis imperfecta) or dentin (dentinogenesis imperfecta) defects could be greatly helped by the availability of functional tooth replacements.
Amelogenesis imperfecta (AI) is a collection of genetic defects manifested by the malformation of dental enamel. One out of every 7,000 to 14,000 children are affected (Backman and Holm, 1986; Chosack et al., 1979; Dummer et al., 1990; and Witkop Jr. and Sauk Jr., 1976). By definition, the disorder must be limited to the dental apparatus and cannot be associated with more generalized defects (Witkop Jr. and Rao, 1971).
Dentinogenesis imperfecta 1 (DGI1) is an autosomal dominant dental disease characterized by abnormal dentin production and mineralization (Xiao et al., 2001). Dentinogenesis imperfecta Sheilds type II (DGI-II) is also an autosomal dominant disorder in which both the primary and permanent teeth are affected. It occurs with an incidence of 1:8,000 live births (Zang et al., 2001).
Recent advances in tissue engineering have demonstrated that organs derived from both epithelial and mesenchymal cells can be fashioned into a pre-determined shape and size and can be provided with a blood supply (Choi et al., 1998; Choi and Vacanti, 1997). Specifically, small pieces (organoid units) of enzymatically digested 6-day-old rat intestine were seeded onto sheets of non-woven polyglycolic acid (PGA) scaffolds and were incubated in culture for various times. Next, they were implanted into the omentum of syngeneic rats. The PGA provided the biodegradable three-dimensional scaffold and implantation into the omentum provided the blood source. The organoid units proliferated and generated larger complex cystic structures that possessed much of the morphology of the mature intestine. A key to the success of the implants was not to delay the in vitro culture time more than is necessary for the organoids to become firmly attached to the scaffold (Choi and Vacanti, 1997). Later, the engineered intestines were further characterized to show that they became phenotypically mature (Choi et al., 1998) and that successful anastomosis occurred between the tissue-engineered intestine and the native small bowel (Kaihara et al., 1999). Since as for the tooth, the intestine is also derived from the interactions of both epithelial and mesenchymal tissues (Haffen et al., 1987), these data provide strong evidence that the dissociated tooth germ may also become fully mature through the techniques of tissue engineering.
A major difference between the tooth and the intestine is that the tooth becomes a mineralized tissue whereas the intestine does not. However, this is not a major technological difficulty since virtually the same tissue-engineering technique used to generated the intestine was also used to engineer mineralized phalanges with joints (Isogai et al., 1999). The phalanges were specifically designed to have a human shape and were shown to possess mature articular cartilage and subchondral bone. Thus, we are generating a tissue-engineered tooth by using techniques similar to those that were used successfully to generate an intestine and phalanges with joints.
The practice of dentistry would be revolutionized, by providing the patient and oral surgeon a means to replace a defective or diseased dentition with a healthy and permanent biological dentition. These studies could yield new insight into the regulation of enamel formation and may provide a means of generating tissue engineered dentin or enamel materials that could be used to repair unhealthy teeth.